Hello!
I am hoping to get some advice/insight about an issue I have run into a few time with quantifying amplicon libraries for running on MiniSeq and NextSeq machine.
I prep my libraries (~400bp) with PCR and then do QC as follows:
- pool samples, qubit
- use qubit concentrations to dilute down to 2nM
- NEB NextQuant qPCR kit with melt curve
- redilute down to 2nM if needed, repeat qPCR to confirm dilutions if I'm extra suspicious
The library concentration of the amplicons I'm currently working with are always drastically underestimated by the qubit. As a result, my 2nM dilution is usually closer to ~5-10nM based on the qPCR results. I run the qPCR in triplicates and the results are always great (sample and control triplicates have close to identical cq values and melt curve is clean). I usually go with the qPCR concentration, but that has led to under-clustering in runs without PhiX. As a note, diversity shouldn't be a big issue with these libraries. Regardless, this has been an issue for over a year and does not seem to come up with other amplicons/libraries.
To try and understand this, I worked with an Illumina "library expert" person and we went through the following theories:
1. bubble products from over-amplification are leading to qubit underestimation. Seems plausible, but there isn't any evidence of bubble products when I've sent libraries to the fragment analyzer. I'd also assume that if the bubble products are to blame, then the qPCR should be trusted which doesn't explain the under-clustering.
2. some sort of structural anomaly with this specific amplicon makes it cluster inefficiently. To address this I've just started using a heat denaturation step prior to loading the cartridge. This doesn't fully explain the discordance between qubit and qPCR values though.
By spiking in 20-50% PhiX and adding a heat denaturation step prior to loading, the libraries are now sequenced beautifully. But I'm still at a loss for why my qubit measurements underestimate so much. I'd love to hear if anyone has had a similar experience or has some idea of what could be causing this library weirdness. Happy to provide more info if needed, thanks!
I am hoping to get some advice/insight about an issue I have run into a few time with quantifying amplicon libraries for running on MiniSeq and NextSeq machine.
I prep my libraries (~400bp) with PCR and then do QC as follows:
- pool samples, qubit
- use qubit concentrations to dilute down to 2nM
- NEB NextQuant qPCR kit with melt curve
- redilute down to 2nM if needed, repeat qPCR to confirm dilutions if I'm extra suspicious
The library concentration of the amplicons I'm currently working with are always drastically underestimated by the qubit. As a result, my 2nM dilution is usually closer to ~5-10nM based on the qPCR results. I run the qPCR in triplicates and the results are always great (sample and control triplicates have close to identical cq values and melt curve is clean). I usually go with the qPCR concentration, but that has led to under-clustering in runs without PhiX. As a note, diversity shouldn't be a big issue with these libraries. Regardless, this has been an issue for over a year and does not seem to come up with other amplicons/libraries.
To try and understand this, I worked with an Illumina "library expert" person and we went through the following theories:
1. bubble products from over-amplification are leading to qubit underestimation. Seems plausible, but there isn't any evidence of bubble products when I've sent libraries to the fragment analyzer. I'd also assume that if the bubble products are to blame, then the qPCR should be trusted which doesn't explain the under-clustering.
2. some sort of structural anomaly with this specific amplicon makes it cluster inefficiently. To address this I've just started using a heat denaturation step prior to loading the cartridge. This doesn't fully explain the discordance between qubit and qPCR values though.
By spiking in 20-50% PhiX and adding a heat denaturation step prior to loading, the libraries are now sequenced beautifully. But I'm still at a loss for why my qubit measurements underestimate so much. I'd love to hear if anyone has had a similar experience or has some idea of what could be causing this library weirdness. Happy to provide more info if needed, thanks!
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